Its Compounds Chlorogenic Acid and Astilbin Inhibit the Activity of �-Amylase and �-Glucosidase Enzymes
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Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 6247306, 12 pages https://doi.org/10.1155/2018/6247306 Research Article Smilax aristolochiifolia Root Extract and Its Compounds Chlorogenic Acid and Astilbin Inhibit the Activity of �-Amylase and �-Glucosidase Enzymes Viridiana Candelaria Pérez-Nájera,1 Janet Alejandra Gutiérrez-Uribe ,2 Marilena Antunes-Ricardo,2 Sergio Hidalgo-Figueroa,3 Carmen Lizette Del-Toro-Sánchez,4 Luis A. Salazar-Olivo ,5 and Eugenia Lugo-Cervantes 6 1 Division´ de Desarrollo Biotecnologico,´ Centro Universitario de la Cienega-Universidad´ de Guadalajara, 47820 Ocotlan,´ Mexico 2TecnologicodeMonterrey,CentrodeBiotecnolog´ ´ıa-FEMSA, 64849 Monterrey, Mexico 3Catedra´ CONACYT, IPICYT/Consorcio de Investigacion,´ Innovacion´ y Desarrollo para las Zonas Aridas,´ 78216 San Luis Potos´ı, Mexico 4Departamento de Investigacion´ y Posgrado en Alimentos, Universidad de Sonora, 83000 Hermosillo, Mexico 5Division´ de Biolog´ıa Molecular, Instituto Potosino de Investigacion´ Cient´ıfca y Tecnologica´ (IPICYT), 78216 San Luis Potos´ı, Mexico 6Unidad de Tecnolog´ıa Alimentaria, Centro de Investigacion´ y Asistencia en Tecnolog´ıa y Diseno˜ del Estado de Jalisco, 44270 Guadalajara, Mexico Correspondence should be addressed to Luis A. Salazar-Olivo; [email protected] and Eugenia Lugo-Cervantes; [email protected] Received 20 February 2018; Revised 12 April 2018; Accepted 23 April 2018; Published 25 June 2018 Academic Editor: Michał Tomczyk Copyright © 2018 Viridiana Candelaria Perez-N´ ajera´ et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Regulating activities of �-amylase and �-glucosidase through the use of specifc inhibitors is a main strategy for controlling type 2 diabetes. Smilax aristolochiifolia root decoctions are traditionally used in Mexico as hypoglycemic and for weight loss, but the active principles and mechanisms underlying such putative metabolic efects are yet unknown. Here, we isolated the major bioactive compounds from a hydroethanolic extract of S. aristolochiifolia root by fast centrifugal partition chromatography and evaluated their efects against pancreatic �-amylase and yeast �-glucosidase. A chlorogenic acid-rich fraction (CAF) inhibited �-amylase activity with an IC50 value of 59.28 �g/mL in an uncompetitive manner and �-glucosidase activity with an IC50 value of 9.27 �g/mL in a noncompetitive mode. Also, an astilbin-rich fraction (ABF) inhibited �-glucosidase activity with an IC50 value of 12.30 �g/mL, in a noncompetitive manner. CAF inhibition �-amylasewasasactiveasacarbosewhilebothCAFandABFwere50-foldmore potent inhibitors of �-glucosidase than acarbose. Te molecular docking results of chlorogenic acid and astilbin with �-amylase and �-glucosidase enzymes correlated with the inhibition mechanisms suggested by enzymatic assays. Our results prove that S. aristolochiifolia roots contain chlorogenic acid and astilbin, which inhibit carbohydrates-hydrolyzing enzymes, suggesting a new mechanism for the hypoglycemic efect reported for this plant. 1. Introduction by abnormally high plasma glucose concentration, resulting from insufcient or inefcient insulin secretion, with alter- Diabetes mellitus is one of the most common chronic diseases ations in carbohydrate, protein, and lipid metabolism. Hyper- in nearly all countries and continues to increase in number glycemia has played a central role in the pathogenesis of and signifcance, as economic development and urbanization complications related to diabetes mellitus, such as retinopa- lead to lifestyles characterized by reduced physical activity thy, cataract, atherosclerosis, neuropathy, nephropathy, and and increased obesity [1]. Diabetes mellitus is characterized impaired wound healing [2]. One therapeutic approach 2 Evidence-Based Complementary and Alternative Medicine for decreasing postprandial hyperglycemia is to reduce the paper no. 4. Ten, ethanol was eliminated by concentration ∘ intestinal absorption of glucose from food, through inhibit- under vacuum (IKA RV 10 digital, Staufen, Germany) at 40 C ∘ ing the intestinal carbohydrate-hydrolyzing enzymes, �- and water by freeze-drying. Dry SAR was stored at −80 C amylase and �-glucosidase. Synthetic drugs such as acarbose, until use. voglibose, and miglitol are widely used as inhibitors of these enzymes in the management of patients with type 2 2.3. Fast Centrifugal Partition Chromatography. Fractions diabetes [3, 4]. However, these inhibitors are reported to cause from SAR were obtained in a preparative fast centrifugal several side efects, such as abdominal distension, fatulence, partition chromatography (FCPC) instrument (Kromaton, meteorism, and diarrhea. Previously studies suggested that Angers,France),withrotorcapacityof1L,operatedindual- the consumption of dietary polyphenols might reduce the mode: 0–57 min in descending mode, and 58–120 min in risk of type 2 diabetes and its complications [5–8]. Terefore, ascending mode at 1000 rpm and a fow rate of 10 mL/min eforts have been directed toward fnding natural and safer using ethyl acetate: water (1 : 1 v/v) as the two-phase solvent �-amylase and �-glucosidase inhibitors, and the search of system, according to preliminary assays (Table S1). SAR such agents in traditional medicinal plants has become more (10g)wasdissolvedin160mLofsolventsystem,fltered,and important [9]. pumped into the rotor. One hundred and twenty fractions Smilax aristolochiifolia Miller (Smilacaceae), popularly were collected and grouped in pools of 10 fractions according known as zarzaparrilla, is widely distributed in Mexico [10] to similarity of their partition coefcient (��)valuesto and commonly employed as root decoctions indicated as facilitate their analysis. A total 12 pools were concentrated to ∘ hypoglycemic [11] and for weight loss [12]. Pharmacological dryness at 45 C under reduced pressure (EZ-2 Plus, Genevac ∘ research has reported hematopoietic [13], hypoglycemic, and Ltd.,UK)andstoredat−20 Cuntiltesting. hypotensiveefects[14]fortherootofS. aristolochiifolia. Although antidiabetic potential has also been reported for 2.4. High Performance Liquid Chromatography Analysis. SAR other Smilax species, mainly of S. china [15,16],theidentityof and its FCPC-obtained fractions were analyzed by HPLC- bioactive compounds responsible for the antidiabetic efects DAD (Agilent Technologies, 1200 Series, Santa Clara, CA) of S. aristolochiifolia as well as their mechanisms of action according to the method described by Becerra-Moreno et al. are yet unknown. Terefore, we aim to identify the major [17] with some modifcations. Te compounds were separated bioactive compounds from S. aristolochiifolia root and to in a Luna 5 U C18,4.6mmID× 250 mm (5 �m) column (Phe- characterize their efects on �-amylase and �-glucosidase nomex,Torrance,CA).Temobilephasewasconstitutedby enzymatic activities. solvent A, HPLC grade water (BDH, Poole, UK) acidifed with 0.1% formic acid (CTR Scientifc, Monterrey, NL, Mex- 2. Materials and Methods ico),andsolventB,HPLCgrademethanol(BDH,Poole,UK), using a gradient at a fow rate of 0.8 mL/min. Te proportion 2.1. Materials. Plants of Smilax aristolochiifolia Miller (in- of the mobile phase was maintained as follows: 0–3 min (B, cluding the roots) were collected in Apazapan, Veracruz, ∘ � �� ∘ � �� 0% to 18%); 3–8 min (B, 18% to 30%); 8–35 min (B, 30% to Mexico (19 19 25.6 Nand9643 17.3 W) in October 2015. 42%); 35–40 min (B, 42% to 48%); 40–45 min (B, 48% to Plant material was authenticated by Dr. M. Chazaro (Biology 60%); 45–50 min (B, 60% to 100%); 50–60 min (B, 100% to Department, Universidad Veracruzana), and a voucher speci- 0%). Chromatograms were obtained at 280 nm, 10 �Lofsam- men (10855) was deposited in the Institute of Ecology herbar- ple was injected, and UV absorption spectra were collected. ium (IE-XAL), Xalapa, Veracruz, Mexico. �-Glucosidase Te results of quantifcation were expressed as chlorogenic (EC 3.2.1.20, from Saccharomyces cerevisiae, 28 U/mg), acar- acid or kaempferol-3-O-glucoside equivalents, based on the bose, �-nitrophenyl-�-D-glucopyranoside (pNPG), porcine calibration curve of the corresponding standards. pancreatic �-amylase (EC 3.2.1.1, type VI-B, from porcine Identifcation of major compounds was carried out by pancreas, ≥10 U/mg), and 3,5-dinitrosalicylic acid reagent liquid chromatography coupled with time-of-fight mass (DNS) were purchased from Sigma-Aldrich Co. (St. Louis, spectrometry (LC/MS-TOF) (1100 Series, Agilent Technolo- MO, USA). Te soluble starch was purchased from Jalmek gies, Santa Clara, CA), using the same chromatographic Cient´ıfca (Monterrey, NL, Mexico). conditions described above. Ionization was carried out using + an electrospray ionization source in positive mode (ESI ) 2.2. Preparation of S. aristolochiifolia Root Extract. Te root of with the following conditions: range for mass scan covered ∘ the plant was dried in the dark at room temperature and the from �/� 140to1000,nitrogengastemperaturesetat350C, dried material was then milled with a ball mill. Preliminary gas fow rate at 11 L/min, nebulizer pressure at 50 psi, 3500 V assays showed that extraction of S. aristolochiifolia roots by capillary voltage, and 50 V in fragmentor. Extracted ion aqueous infusion or hydroethanolic maceration gives rise to chromatograms were obtained by considering the exact mass the same profle of elution